Intestinal mucosal barrier function in health and disease 2009.pdf · mucosa-associated lymphoid tissue (MALT) is exposed to and interacts with the external environment. The magni

Complex multicellular organisms interface with their external environments at multiple sites, including mucosae of the airways, oral cavity, digestive tract and genitourinary tract, and the skin. Although the skin is the most visible site of interface, the combined area of the mucosal surfaces is much greater than that of the skin. Mucosae are also the primary sites at which the mucosa-associated lymphoid tissue (MALT) is exposed to and interacts with the external environment. The magni-tude of these interactions is greatest in the gastrointesti-nal tract, which is the largest mucosal surface and is also in continuous contact with dietary antigens and diverse microorganisms. Thus, mucosal surfaces, particularly in the intestines, are crucial sites of innate and adaptive immune regulation.

A central mediator of interactions between MALT and the external environment, including the intesti-nal lumen, is the epithelium that covers the mucosa. Epithelial cells establish and maintain the barrier, which in the skin is a tight, although not impermeant, seal. By contrast, most mucosal epithelial cells form leaky barriers. This is necessary to support universal functions that include fluid exchange, which occurs at nearly all mucosal surfaces, as well as essential tissue-specific functions. Thus, the precise permea-bility characteristics of each barrier type differ based on the functions supported. For example, the ion-selective properties of the barrier vary considerably along the length of a nephron to promote or restrict transport of specific ions in each segment1. Similarly, the ability of tight junctions to discriminate between and restrict passage of solutes based on size (a characteristic known

as size selectivity) varies with location in the intestine, as permeability to larger solutes decreases from the crypt to the villus2. In addition to these fixed differences, mucosal permeability of many tissues is adaptable and may be regulated in response to extracellular stimuli, such as nutrients, cytokines and bacteria.

Recent advances have uncovered some of the mech-anisms by which physiological and immunological stimuli affect cellular and extracellular components of the intestinal barrier. In this article I review our current understanding of the mechanisms that regulate intesti-nal barrier integrity, and discuss the hypothesis that the mucosal barrier can shape pro-inflammatory and immuno-regulatory responses in the context of homeostasis and disease.

Although they may affect barrier function, den-dritic cells (DCs), which extend dendritic processes across the tight junctions in the distal small intestine3,4, and intraepithelial lymphocytes are not discussed here. In addition, the functions of M cells, which are spe-cialized epithelial cells that deliver antigens directly to intra epithelial lymphocytes and to subepithelial lymphoid tissues by transepithelial vesicular transport from the gut lumen, are reviewed elsewhere5. Finally, detailed analyses of the interactions between epithelial cells and luminal microorganisms, as well as the intri-cacies of mucosal immune regulation, have also been recently reviewed elsewhere6,7. This Review therefore focuses on the roles of the epithelial cell barrier in health and disease, with particular emphasis on the junctional complexes between intestinal epithelial cells, which have a crucial role in barrier regulation.

Department of Pathology, The University of Chicago, 5841 South Maryland, MC 1089, Chicago, Illinois 60637, USA.e-mail: [email protected]:10.1038/nri2653

Mucosa-associated lymphoid tissue(MALT). The collections of B cells, T cells, plasma cells, macrophages and other antigen-presenting cells found in the mucosal linings of organs including the gastrointestinal tract, lungs, salivary glands and conjuctiva.

Tight junctionAlso known as the zonula occludens, this is a site of close apposition of adjacent epithelial cell membranes — kiss points — that create a barrier against the free diffusion of water and solutes.

Intestinal mucosal barrier function in health and diseaseJerrold R. Turner

Abstract | Mucosal surfaces are lined by epithelial cells. These cells establish a barrier between sometimes hostile external environments and the internal milieu. However, mucosae are also responsible for nutrient absorption and waste secretion, which require a selectively permeable barrier. These functions place the mucosal epithelium at the centre of interactions between the mucosal immune system and luminal contents, including dietary antigens and microbial products. Recent advances have uncovered mechanisms by which the intestinal mucosal barrier is regulated in response to physiological and immunological stimuli. Here I discuss these discoveries along with evidence that this regulation shapes mucosal immune responses in the gut and, when dysfunctional, may contribute to disease.

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nATuRE REvIEwS | Immunology voLuME 9 | novEMBER 2009 | 799

© 2009 Macmillan Publishers Limited. All rights reserved

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E-cadherin

F-actinMyosinOccludin

Desmoglein

Keratin

Desmoplakin

Desmocollin

β-catenin

α-catenin 1

a b

ClaudinZO1

MLCK

Tight junction

Desmosome

Adherensjunction

Nature Reviews | Immunology

Microvilli

Mucus

Goblet cell

Unstirred layer

Epithelial cells

Intraepithelial lymphocyte

Basement membrane

Plasma cell

Apicaljunctional complex

Lamina proprialymphocyte

MucinsA family of heavily glycosylated proteins that are secreted as large aggregates by mucous epithelial cells.

Unstirred layerA thin layer of fluid at epithelial cell surfaces that is separated from the mixing forces created by luminal flow and, in the intestine, peristalsis.

Coeliac diseaseA chronic inflammatory condition of the upper small intestine in humans that is caused by immunological hypersensitivity to the α-gliadin component of wheat gluten. It can cause severe villous atrophy, which can lead to malabsorption and malnutrition if gluten-containing foods are not removed from the diet.

Anatomy of mucosal barriersExtracellular components of the barrier. Most mucosal surfaces are covered by a hydrated gel formed by mucins (FIG. 1a). Mucins are secreted by specialized epithelial cells, such as gastric foveolar mucous cells and intestinal goblet cells, and create a barrier that prevents large par-ticles, including most bacteria, from directly contacting the epithelial cell layer8. The importance of mucus gel hydration is shown by cystic fibrosis, in which the pro-duction of hyperviscous mucus contributes to pulmo-nary, pancreatic and intestinal disease9. Defective mucus production has also been reported in various immune-mediated diseases, and spontaneous colitis develops in mice that lack specific mucin genes10.

Although small molecules pass through the heavily glycosylated mucus layer with relative ease, bulk fluid flow is limited and thereby contributes to the develop-ment of an unstirred layer of fluid at the epithelial cell sur-face. As the unstirred layer is protected from convective mixing forces, the diffusion of ions and small solutes is slowed. In the stomach, this property of the unstirred layer works with epithelial cell bicarbonate secretion to main-tain a zone of relative alkalinity at the mucosal surface11. The unstirred layer of the small intestine slows nutrient

absorption by reducing the rate at which nutrients reach the transporting protein-rich microvillus brush border, but may also contribute to absorption by limiting the extent to which small nutrients released by the activities of brush border digestive enzymes are lost by diffusion into the lumen. unstirred layer defects have not been linked to specific diseases. However, it is interesting to note that increased unstirred layer thickness has been reported in coeliac disease12, in which it may contribute to nutrient malabsorption.

Cellular components of the mucosal barrier. The pri-mary responsibility for mucosal barrier function resides with the epithelial cell plasma membrane, which is impermeable to most hydrophilic solutes in the absence of specific transporters. Accordingly, direct epithelial cell damage, such as that induced by mucosal irritants or cytotoxic agents, including some drugs used for can-cer chemotherapy, results in a marked loss of barrier function. However, in the presence of an intact epithe-lial cell layer, the paracellular pathway between cells must be sealed. This function is mediated by the apical junc-tional complex, which is composed of the tight junction and subjacent adherens junction (FIG. 1b). Both tight and

Figure 1 | Anatomy of the mucosal barrier. a | The human intestinal mucosa is composed of a simple layer of columnar epithelial cells, as well as the underlying lamina propria and muscular mucosa. Goblet cells, which synthesize and release mucin, as well as other differentiated epithelial cell types, are present. The unstirred layer, which cannot be seen histologically, is located immediately above the epithelial cells. The tight junction, a component of the apical junctional complex, seals the paracellular space between epithelial cells. Intraepithelial lymphocytes are located above the basement membrane, but are subjacent to the tight junction. The lamina propria is located beneath the basement membrane and contains immune cells, including macrophages, dendritic cells, plasma cells, lamina propria lymphocytes and, in some cases, neutrophils. b | An electron micrograph and corresponding line drawing of the junctional complex of an intestinal epithelial cell. Just below the base of the microvilli, the plasma membranes of adjacent cells seem to fuse at the tight junction, where claudins, zonula occludens 1 (ZO1), occludin and F‑actin interact. E‑cadherin, α‑catenin 1, β‑catenin, catenin δ1 (also known as p120 catenin; not shown) and F‑actin interact to form the adherens junction. Myosin light chain kinase (MLCK) is associated with the perijunctional actomyosin ring. Desmosomes, which are located beneath the apical junctional complex, are formed by interactions between desmoglein, desmocollin, desmoplakin and keratin filaments.

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Paracellular pathwayThe route of transepithelial transport that involves passive movement through the space between adjacent cells.

Adherens junctionAlso known as the zonula adherens, this junction is immediately subjacent to the tight junction and requires the activity of lineage-specific Ca2+-dependent adhesion proteins, termed cadherins.

DesmosomeAn adhesive junction that connects adjacent epithelial cells. These junctions are composed of multiple protein subunits and are the points where keratin filaments attach to the plasma membrane.

ClaudinFrom the Latin claudere, meaning ‘to close’, members of this family of transmembrane proteins are variably expressed by specific epithelial cell types and thereby contribute to the unique barrier properties of different tissues.

OccludinThe first transmembrane tight junction protein identified. The function of occludin remains controversial but it is likely to have roles in barrier regulation and tumour suppression. It also serves as a cofactor in hepatitis C virus entry.

Zonula occludens 1A peripheral membrane, or plaque, protein containing multiple protein interaction domains that, along with the related protein zonula occludens 2, is required for tight junction assembly.

Transcellular pathwayThe route of transepithelial transport that involves active or passive movement across cell membranes, usually as a result of the action of specific transport channels.

Transepithelial transportThe sum of transport through the transcellular and paracellular pathways.

adherens junctions are supported by a dense perijunc-tional ring of actin and myosin that, as discussed below, can regulate barrier function.

As implied by the name, the adherens junctions, along with desmosomes, provide the strong adhesive bonds that maintain cellular proximity and are also a site of intercellular communication. Loss of adherens junctions results in disruption of cell–cell and cell–matrix contacts, ineffective epithelial cell polariza-tion and differentiation, and premature apoptosis13. Adherens junctions are composed of cadherins, a family of transmembrane proteins that form strong, homotypic interactions with molecules on adjacent cells. The cyto-plasmic tail of the epithelial cadherin, E-cadherin (also known as cadherin-1), interacts directly with catenin δ1 (also known as p120 catenin) and β-catenin. In turn, β-catenin binds to α-catenin 1, which regulates local actin assembly and contributes to development of the perijunctional actomyosin ring.

Adherens junctions are required for assembly of the tight junction, which seals the paracellular space. Tight junctions are multi-protein complexes composed of transmembrane proteins, peripheral membrane (scaf-folding) proteins and regulatory molecules that include kinases. The most important of the transmembrane proteins are members of the claudin family, which define several aspects of tight junction permeability, as dis-cussed below. Claudins are expressed in a tissue-specific manner, and mutation or deletion of individual family members can have profound effects on organ function. The role of occludin, a transmembrane tight junction protein that interacts directly with claudins and actin, is less well understood. Peripheral membrane proteins, such as zonula occludens 1 (Zo1) and Zo2, are crucial to tight junction assembly and maintenance, partly owing to the fact that these proteins include multiple domains for interaction with other proteins, including claudins, occludin and actin.

The tight junction limits solute flux along the para-cellular pathway, which is typically more permeable than the transcellular pathway. The tight junction is, there-fore, the rate-limiting step in transepithelial transport and the principal determinant of mucosal permeability. Thus, it is important to understand the specific barrier properties of the tight junction, which can be defined in terms of size selectivity and charge selectivity.

At least two routes allow transport across the tight junction, and emerging data suggest that the relative contributions of these types of paracellular transport may be regulated independently2,14,15. one route, the leak pathway, allows paracellular transport of large solutes, including limited flux of proteins and bacterial lipopolysaccharides14,15. Although the size at which par-ticles are excluded from the leak pathway has not been precisely defined, it is clear that materials as large as whole bacteria cannot pass. As might be expected for a route that allows large solutes to cross, the leak path-way does not show charge selectivity. Flux across the leak pathway may be increased by cytokines, including interferon-γ (IFnγ) in vitro and tumour necrosis factor (TnF) in vitro and in vivo15–17.

A second pathway is characterized by small pores that are thought to be defined by tight junction-associated claudin proteins, which are also primary determinants of charge selectivity18–20. These pores have a radius that excludes molecules larger than 4 Å14,15. Expression of spe-cific claudins varies between organs and even within dif-ferent regions of a single organ and, as detailed below, can be modified by external stimuli, such as cytokines. Thus, tight junctions show both size selectivity and charge selec-tivity, and these properties may be regulated individually or jointly by physiological or pathophysiological stimuli.

Interdependence of transport routesTransepithelial transport requires a selectively permeable barrier. The vectorial nature of transcellular transport, which is required for effective absorption and secre-tion, generates a transepithelial concentration gradient. Transepithelial transport would, therefore, be ineffective without a tight junction barrier, as diffusion would allow equalization of concentrations on both sides of the epith-elium. Thus, active transcellular transport depends on the presence of an intact tight junction barrier. The selective permeability of the tight junction barrier also allows trans-epithelial gradients to drive passive paracellular transport of ions and water17,20. For example, claudin-16, which is necessary for tight junction cation selectivity, is mutated in the human disease familial hypomagnesaemia with hyper-calciuria and nephro calcinosis20. Paracellular absorption of Mg2+ and Ca2+ in the thick ascending limb of Henle requires a cation-selective tight junction barrier, the absence of which results in urinary loss of Mg2+ and Ca2+ (ReF. 20). Similarly, apical na+–H+-exchange protein 3 (nHE3) is required for transcellular na+ absorption in the renal tubule and intestine, and also contributes to the trans-epithelial na+ gradient that drives paracellular water absorption (BOX 1). The tight junction barrier to para-cellular na+ efflux prevents dissipation of the gradient established by nHE3-mediated transcellular transport.

Tight junction barrier regulation modifies absorption. In addition to providing the driving force for paracel-lular transport, transcellular transport can activate intra-cellular signalling events that regulate the tight junction barrier. The best studied physiological example of this is the increased intestinal paracellular permeability that is induced by apical na+–nutrient co-transport21. This allows passive paracellular absorption of nutrients and water to amplify transcellular nutrient absorption, partic-ularly when high luminal nutrient concentrations exceed the capacity of apical na+–nutrient co-transporters22. ultrastructural analyses of intestinal epithelial cells during na+–nutrient co-transport revealed condensation of the perijunctional actomyosin ring, suggesting that cytoskel-etal contraction could be involved in this physiological process21. Subsequent studies confirmed this hypothesis and showed that the Ca2+–calmodulin-dependent serine–threonine protein kinase myosin light chain kinase (MLCK) is essential for na+–nutrient co-transport-induced tight junction regulation23,24. Moreover, MLCK activation alone is sufficient to increase tight junction perme ability, both in vitro and in vivo25,26. The mechanisms that mediate this

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http://www.uniprot.org/uniprot/P12830

http://www.uniprot.org/uniprot/O60716



http://www.uniprot.org/uniprot/Q07157

http://www.uniprot.org/uniprot/Q9UDY2



http://www.uniprot.org/uniprot/Q9Y5I7



Thick ascending limb of HenleThe portion of the nephron just proximal to the distal tubule. This is a site of active Na+, K+ and Cl– reabsorption, which generates an electrochemical gradient that drives paracellular reabsorption of Mg2+ and Ca2+.

Myosin light chain kinaseThe Ca2+–calmodulin-dependent kinase that phosphorylates myosin II regulatory light chain at serine 19 and threonine 18 to activate myosin ATPase.

MLCK-dependent tight junction regulation have been studied in detail owing to their contributions to nutri-ent and water absorption under normal physiological conditions and to the diarrhoea associated with acute barrier loss (BOX 1).

Barrier regulation by immune stimuli The ability of cytokines, such as TnF and IFnγ, to regulate the function of the tight junction barrier was first described 20 years ago27. Since then, increased tight junction protein transcription, vesicular removal of pro-teins from the tight junction, tight junction protein deg-radation, kinase activation and cytoskeletal modulation have all been proposed to mediate cytokine-induced loss of tight junction barrier function. Although extensive apoptosis of epithelial cells may also cause barrier loss, the relevance of single-cell apoptosis to barrier dysfunc-tion remains controversial owing to differing results in diverse experimental systems.

Tight junction regulation through the cytoskeleton. TnF and IFnγ modify tight junction barrier function in intestinal27,28, renal29, pulmonary30 and salivary gland31 epithelia as well as between endothelial cells32. The effects of TnF on barrier integrity have been best studied in the gut, where this cytokine has a central role in many diseases associated with intestinal epith elial barrier dysfunction, including inflammatory bowel disease33, intestinal ischaemia28,34 and graft-versus- host disease35. For example, although the effect of therapy

with TnF-specific antibodies may be largely due to the overall reduction in inflammation, it is notable that this treatment corrects barrier dysfunction in patients with Crohn’s disease36. Increased mucosal TnF production may also contribute to increased intestinal permeabil-ity and susceptibility to colitis in mice with defective mucin biosynthesis10,37.

MLCK has been shown to have a central role in TnF-induced epithelial and endothelial barrier dysregulation, both in vitro and in vivo16,38–41. Similar to na+–nutrient co-transport, TnF-induced MLCK activation seems to increase paracellular flux through the leak pathway16–17,23. This MLCK activation occurs as a result of increased enzymatic activity and increased MLCK transcription and translation, both in vitro and in vivo16,40,42. Similarly, MLCK expression and activity are increased in intesti-nal epithelial cells of patients with inflammatory bowel disease43. The degree to which MLCK expression and activity are increased correlates with local disease activ-ity, suggesting that these processes may be regulated by local cytokine signalling in these patients43. MLCK is also a fundamental intermediate in barrier dysfunction induced by the TnF family member LIGHT (also known as TnFSF14)17,44, interleukin-1β (IL-1β)45, enteropatho-genic Escherichia coli infection39, Helicobacter pylori infection46, giardiasis47, lipopolysaccharide48,49 and the ethanol metabolite acetaldehyde50. Thus, MLCK activa-tion can be viewed as a common final pathway of acute tight junction regulation in response to a broad range of immune and infectious stimuli.


Nodiarrhoea

H2O

Na+

Na+

a Normal homeostasisH2O

Na+

b Barrier disruption only

Milddiarrhoea

H2O

Na+

Na+

c Na+ malabsorption only

H2O

H2O

Na+

Na+

d Na+ malabsorption and barrier disruption

Nodiarrhoea

Copiousdiarrhoea

Na+

Box 1 | Coordination of transcellular and paracellular transport

The signal transduction pathway that enhances paracellular permeability following initiation of Na+–glucose co-transport has been characterized and includes activation of mitogen-activated protein kinase cascades, trafficking of Na+–H+-exchange protein 3 (NHE3) to the apical membrane and myosin light chain kinase (MLCK) activation23–24,107. Increased NHE3 activity at the apical membrane enhances Na+ absorption, which (along with the Na+ absorbed as a result of Na+–glucose co-transport) increases the transcellular Na+ gradient and promotes paracellular water absorption (see the figure, part a). The MLCK-dependent increase in tight junction permeability enhances paracellular absorption of water and small solutes, such as glucose, that are concentrated in the unstirred layer. This might contribute to the observation that rehydration after infectious diarrhoea, or even exercise, is more effective when oral rehydration solutions contain Na+ and carbohydrates108 (see the figure, part b). By contrast, tumour necrosis factor activates both protein kinase Cα (PKCα) and MLCK to cause diarrhoea. NHE3 inhibition, mediated by PKCα, reduces the transcellular Na+ gradient that normally drives water absorption (see the figure, part c). This synergizes with MLCK- dependent increases in tight junction permeability to allow water to flow into the lumen, thereby causing large-volume diarrhoea17 (see the figure, part d).

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a b

c d

CaveolaeSpecialized flask-shaped invaginations of the plasma membrane that contain the protein caveolin-1 and cholesterol. These proteins mediate uptake of some extracellular materials and are involved in cell signalling.

Although it is clear that MLCK phosphorylates myosin II regulatory light chain (MLC) within the peri-junctional actomyosin ring to activate myosin ATPase activity, the subsequent molecular events that cause increased permeability are poorly defined. However, recent work suggests that Zo1, which interacts directly with actin, occludin, claudins and other proteins, may be an essential effector of perijunctional actomyosin ring-mediated tight junction regulation25,51,52. Endocytic removal of the transmembrane protein occludin from the tight junction is also common in actomyosin-dependent, cytokine-mediated tight junction regulation38,53 (FIG. 2). In vitro studies of tight junction regulation induced by LIGHT suggest that occludin endocytosis occurs via caveolae and that inhibition of this process can prevent loss of barrier integrity despite MLCK activa-tion44. Caveolar endocytosis of occludin has also been associated with loss of epithelial and endothelial tight junction barrier function in response to actin disrup-tion54 and chemokine signalling55, respectively, in vitro. However, other mechanisms of occludin removal may also be involved as in vitro studies have shown that IFnγ-induced occludin internalization is mediated by myosin ATPase-dependent macropinocytosis53,56, and occludin cleavage may even modify the barrier to enhance transepithelial migration of inflamma-tory cells in the lung57. nevertheless, further work is necessary to define the contributions of occludin and endocytosis to in vivo tight junction regulation. This is particularly important as, despite numerous in vitro studies demonstrating a role for occludin in tight junction function, intestinal barrier function is intact in occludin-deficient mice58. Future analyses of the response of occludin-deficient mice to stress, with particular reference to intestinal tight junction

function, as well as studies of potential compensatory changes in other, perhaps not yet discovered, proteins that allow these mice to avoid intestinal disease will be of great interest.

Although MLCK activation is clearly important, it is not the only means of cytoskeletal tight junction regulation. other mediators include myosin ATPase51, the activity of which is regulated by MLC phosphory-lation59–60; members of the Rho kinase family41,51,53,61, which can both phosphorylate MLC directly 59 and inhibit MLC phosphatase62; and AMP-activated protein kinase52, which is activated during stress and can also directly phosphorylate MLC63. Moreover, Rho kinases and AMP-activated protein kinase each have diverse effects that are separate from myosin function, and it is likely that at least some of these contribute to tight junction regulation.

Tight junction permeability and claudin expression. As discussed above, regulation of perijunctional acto-myosin provides a means of rapidly and reversibly regulating the paracellular leak pathway. By contrast, synthesis and trafficking of claudin proteins provides a means of regulating tight junction pores over longer periods. The perijunctional actomyosin ring does not seem to be directly involved in this mechanism of bar-rier regulation, which is consistent with the fact that claudins do not interact with actin directly. Expression of specific claudin proteins changes during develop-ment, differentiation and disease and in response to stressors — including cytokines — in intestinal64, renal65 and alveolar66 epithelial cells. In addition to affecting barrier function, altered patterns of claudin expression may have other consequences. For example, claudin proteins have been associated with control of cell and organ growth. It may be, therefore, that some changes in claudin protein expression enhance cell proliferation and regeneration, as might be necessary to compensate for cell loss in colitis. This may explain the increased claudin-1 expression by intestinal epithelial cells of patients with inflammatory bowel disease67. However, claudin-1 has also been shown to enhance neoplastic transformation, tumour growth and metastasis in exper-imental models68. Thus, changes in claudin expression may have undesired consequences.

one common change in claudin expression that directly affects barrier function is the increased claudin-2 expression by intestinal epithelial cells in animal models of colitis (FIG. 2) and patients with inflammatory bowel disease64. Consistent with this, IL-13 and IL-17, which are increased in the mucosa of patients with colitis64,69, reduce barrier function and increase claudin-2 expres-sion in cultured intestinal epithelium monolayers64,70. In vitro studies have shown that claudin-2 expression increases the number of pores that allow paracellular flux of cations, predominantly na+, and small mole-cules with radii less than 4 Å14,71, and recent analyses have provided new insight into the structure of these pores72. However, it is not clear whether increased claudin-2 expression contributes to disease progres-sion or, as proposed above for claudin-1, is an adaptive

Figure 2 | Differential effects of cytokines on tight junction structure and function. a | Occludin is normally concentrated at the tight junction (arrow) in jejunal villus epithelium. The perijunctional actomyosin ring (red) and nuclei (blue) are shown for reference. b | Exogenous tumour necrosis factor increases myosin light chain kinase activity, which causes perijunctional myosin II regulatory light chain phosphorylation and triggers occludin (green) endocytosis (arrow). This increases flux across the tight junction leak pathway and enhances paracellular permeability to large solutes. c | Claudin‑2 expression (green) is limited to crypt epithelial cells; it is not expressed by epithelial cells at the mucosal surface in the normal colon. F‑actin (red) and nuclei (blue) are shown for reference. d | Interleukin‑13 can stimulate claudin‑2 expression (green) in surface epithelial cells (arrows). This increases flux across small tight junction pores, thereby enhancing paracellular cation permeability.

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SAMP1/yit miceAn outbred mouse strain that spontaneously develops a chronic intestinal inflammation similar to human Crohn’s disease.

response that promotes homeostasis. nevertheless, the consistency of intestinal epithelial claudin-2 upregula-tion in disease suggests that this should be a subject of further study.

Barrier function and immunityBarrier loss is associated with disease risk. A large body of circumstantial evidence suggests that intestinal bar-rier dysfunction is associated with the pathogenesis of Crohn’s disease. This includes the observation that a sub-set of first-degree relatives of patients with Crohn’s dis-ease has increased intestinal permeability despite being completely healthy73. Interestingly, genetic analyses have linked increased intestinal permeability in these healthy relatives to the Crohn’s disease-associated frameshift

insertion at nucleotide 3020 of the gene encoding the cytoplasmic sensor nucleotide-binding oligomerization domain-containing 2 (NOD2)74 (TABLe 1), and the same NOD2 mutation has been associated with decreased IL-10 production by peripheral blood mononuclear cells75. Thus, one explanation for the increased intes-tinal permeability observed in some of these healthy relatives is that they have subclinical mucosal immune activation, perhaps with increased TnF production, that leads to barrier dysfunction without overt disease. This hypothesis would also explain why IL-10-deficient and SAMP1/yit mice, which develop spontaneous colitis and enteritis, respectively, show increased intestinal per-meability before disease onset76,77. The suggestion that permeability is merely a sensitive indicator of mucosal

Table 1 | Barrier defects associated with intestinal disease

Disease or model Cause of barrier defect

Timing of barrier defect

Effects on tight junction proteins and cytoskeleton

Role of commensal microorganisms

Refs

Crohn’s disease Unknown, associated with a frameshift insertion at nucleotide 3020 of NOD2

Before clinical onset and before clinical relapse

Claudin‑2 upregulation, MLCK activation and occludin downregulation

Antibiotics can be helpful in maintaining remission

43,64,109

Ulcerative colitis Unknown Not well studied Claudin‑2 upregulation, MLCK activation and occludin downregulation

Not defined 43,64,109

IL‑10‑deficient mice Immune signalling (IL‑10 deficiency)

Before clinical onset Not defined Eradication of microorganisms prevents disease

76,97

Coeliac disease Not well studied Not well studied Not defined No demonstrated role 110,111

Systemic T cell activation

MLCK activation Associated with acute diarrhoea

MLCK activation and occludin endocytosis

Not defined 38

CD4+CD45RBhi T cell adoptive transfer model of colitis

Cytokine release and epithelial cell damage

With clinical onset MLCK activation, claudin‑2 upregulation and occludin endocytosis

Eradication of microorganisms reduces severity

112–114

SAMP1/yit mice Unknown Before clinical onset Claudin‑2 upregulation and occludin downregulation

Not defined 77

DSS‑induced colitis Epithelial cell damage After DSS treatment but before clinical onset

Not defined Eradication of microorganisms exacerbates disease

80

TNBS‑induced colitis Immune signalling Not well studied Claudin‑18 upregulation Probiotics can reduce disease severity

92

Mucin‑2‑deficient mice

Not well studied Not well studied Not defined Not defined 10,37

MDR1A‑deficent mice

Unknown, follows increased CCL2 production

After immune cell activation

Reduced occludin phosphorylation

Increased epithelial cell response to LPS precedes disease

115,116

JAM‑A‑deficient mice

JAM‑A deficiency Not well studied JAM‑A deficiency Not defined 89,90

Clostridium difficile‑induced colitis

Actomyosin disruption and glucosylation of Rho proteins

With release of toxin and disease onset

Loss of ZO1 and ZO2 Antibiotics predispose to disease

117

EPEC infection Type III secretion (of bacterial proteins)

After infection MLCK activation and occludin endocytosis

Not defined 118

Graft‑versus‑host disease

Associated with elevated TNF production

After clinical onset Not defined Eradication of microorganisms limits disease

35

CCL2, CC‑chemokine ligand 2; DSS, dextran‑sulphate sodium; EPEC, enteropathogenic Escherichia coli; IL‑10, interleukin‑10; JAM‑A, junctional adhesion molecule‑A; LPS, lipopolysaccharide; MDR1A, multidrug resistance protein 1a; MLCK, myosin light chain kinase; NOD, nucleotide‑binding oligomerization domain; TNF, tumour necrosis factor; TNBS, trinitrobenzene sulphonic acid; ZO, zonula occludens.

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CD4+CD45RBhi T cell adoptive transfer colitis modelA well-characterized model of chronic colitis induced by transfer of CD4+CD45RBhi (naive) T cells from healthy wild-type mice into immunodeficient syngeneic recipients.

immune activation would also explain the observa-tion that, during clinical remission, increased intestinal permeability is a predictor of relapse in patients with Crohn’s disease78. However, barrier dysfunction must also be regarded as a potential contributor to disease progression.

The classic experiment showing a role for epithelial cell function in maintaining mucosal immune homeo-stasis used chimeric mice in which dominant-negative cadherin was expressed in some intestinal epithelial stem cells, resulting in disruption of the adherens junctions13. This led to profound epithelial cell defects that included incomplete polarization, brush border and actin cytoskel-etal disruption, accelerated crypt–villus migration and premature apoptosis13. Moreover, the mucus layer over-lying epithelial cells expressing dominant-negative cad-herin was disrupted and numerous adherent bacteria were present at these sites13. Patchy, transmural enteritis developed in these mice and was limited to regions where epithelial cells expressed dominant-negative cadherin79. In addition, epithelial cell dysplasia was occasionally present in these areas79. This study has often been cited as evidence that tight junction barrier loss is sufficient to cause inflammatory bowel disease and, although tight junctions were not specifically examined, they were likely to be defective. However, although this important study shows that apical junctional complex disruption causes enteritis, the broad range of epithelial cell defects induced makes it impossible to draw conclusions regarding the specific contributions of the tight junction to homeostasis or disease. Similarly, although dextran-sulphate sodium (DSS)-induced colitis is associated with increased intes-tinal permeability, this is the result of widespread epithe-lial cell damage80 rather than targeted dysfunction of the tight junction barrier. Altered sensitivity of genetically modified mice to DSS must therefore be viewed in the context of epithelial cell injury and repair81–83 and cannot be interpreted as a function of disrupted tight junction permeability alone.

Isolated barrier loss is insufficient to cause disease. one study has reported an in vivo model of barrier dys-function induced by direct activation of endogenous tight junction regulatory mechanisms26. Although no overt developmental defects or disease developed in mice in which intestinal epithelial cells transgenically expressed constitutively active MLCK, increased intes-tinal tight junction permeability was observed26. This increase in paracellular permeability was quantitatively similar to that induced by activation of na+–glucose co-transport and was not an indiscriminate loss of barrier function26, as occurs with exposure to DSS or dominant-negative cadherin expression. In addition, MLCK inhibition normalized MLC phosphorylation in epithelial cells and paracellular permeability26, sug-gesting that compensatory changes in pathways that regulate MLC phosphorylation or barrier function were not induced. Moreover, growth of these mice was normal, and intestinal morphology, enterocyte struc-ture, tight junction and adherens junction organiza-tion, actomyosin and brush border architecture, and

epithelial proliferation, migration and apoptosis were all similar to that observed in wild-type mice26. This suggests that the effects of constitutively active MLCK expression in these mice were specific to its effect on the tight junction. Thus, consistent with the presence of barrier dysfunction in healthy relatives of patients with Crohn’s disease, these mice provide evidence to suggest that increased tight junction permeability in the absence of more extensive epithelial cell dysfunc-tion is insufficient to cause intestinal disease. Further analysis of mice expressing constitutively active MLCK did, however, provide evidence of mucosal immune cell activation, including increased numbers of lamina pro-pria T cells, enhanced mucosal IFnγ, TnF and IL-10 transcription and repositioning of CD11c+ DCs to the superficial lamina propria26. Thus, despite being insuf-ficient to cause disease, chronic increases in intestinal permeability as a result of continuous activation of a physiological pathway of tight junction regulation does lead to mucosal immune cell activation.

Because a subset of healthy first-degree relatives of patients with Crohn’s disease will ultimately develop the disease, and because a similar fraction of these healthy relatives have increased intestinal permeability, it has been suggested that tight junction barrier dysfunction is one factor that contributes to the development of inflammatory bowel disease84. This hypothesis is consist-ent with a case report documenting increased intestinal permeability 8 years prior to disease onset in a healthy relative of a patient with Crohn’s disease85. However, given that the subject of the case report had two first-degree relatives with Crohn’s disease and was, therefore, at increased risk of developing the disease regardless of intestinal permeability measures, this report does not provide evidence that barrier dysfunction itself is related to disease onset.

The relationship between barrier dysfunction and disease has been assessed using mice with a constitutively active MLCK transgene, which have increases in permea-bility that are quantitatively and qualitatively similar to those in healthy relatives of patients with Crohn’s disease. Recombination-activating gene 1-knockout mice (which lack mature B and T cells) expressing constitutively active MLCK, as well as littermates that did not express constitutively active MLCK, received CD4+CD45RBhi naive T cells from wild-type mice (the CD4+CD45RBhi T cell adoptive transfer colitis model). Both groups of mice developed colitis. However, the disease developed more rapidly and was more severe, in clinical, biochemical and histological terms, in the mice that expressed constitu-tively active MLCK26. Although one could argue that this accelerated disease progression was due to effects of constitutively active MLCK expression beyond tight junction regulation, other epithelial cell defects were not apparent in these mice (see above), perhaps because con-stitutively active MLCK targets an endogenous signalling pathway. Thus, targeted increases in intestinal epithelial tight junction permeability through constitutive activa-tion of the pathophysiologically relevant MLCK pathway are sufficient to accelerate the onset and enhance the severity of immune-mediated colitis.

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Latency-associated peptideA small peptide derived from the N-terminal region of the TGFβ precursor protein; it can modulate TGFβ signalling.

Conversely, the data above also suggest that inhibi-tion of MLCK, which is sufficient to reverse acute TnF-induced barrier loss38, may have therapeutic benefit in immune-mediated colitis. This notion is supported by a preliminary in vivo report using mice with a knock-out of non-muscle MLCK86. However, as non-muscle MLCK has many functions, including an essential role in neutrophil transendothelial migration87, the results of this report may reflect changes beyond tight junc-tion barrier preservation. Another recent study showed that a peptide capable of enhancing small-intestinal barrier function reduced the extent of mucosal inflam-mation in IL-10-deficient mice88. However, this result must be interpreted with caution, as the peptide used (which is thought to antagonize the putative extra-cellular pathway signalling molecule zonulin) activates an undefined regulatory pathway and the overall physi-ology of peptide-treated mice has not been examined in detail. Thus, although the finding is intriguing, further investigation is necessary to determine whether barrier preservation, by MLCK inhibition or other means, can prevent or reverse intestinal disease.

Two studies have recently reported that junctional adhesion molecule-A (JAM-A)-deficient mice have reduced intestinal barrier function and increased rates of intestinal epithelial cell proliferation and apopto-sis89,90. These mice also have intestinal mucosal immune cell activation as defined by increased numbers of neutrophils in the intestinal mucosa89,90. In addition, one study reported an increase in the number of dis-tal-colonic lymphoid aggregates in JAM-A-deficient mice90, although neither lymphoid aggregates nor enhanced mucosal cytokine expression was detected in a subsequent analysis89. However, as JAM-A is widely expressed by epithelial cells, endothelial cells, platelets, antigen-presenting cells, circulating neutrophils, mono-cytes, lymphocytes and platelets, and as the mice studied were not epithelial cell-specific knockouts, it is not clear whether the observed effects were due to altered tight junction integrity, changes in epithelial cell shape91, an increased rate of epithelial cell apoptosis90 or trapping of neutrophils within mucosal vessels owing to the loss of endothelial cell-expressed JAM-A. nevertheless, it is intriguing that JAM-A-deficient mice showed increased sensitivity to DSS-induced colitis89,90. By contrast, the sensitivity of mice with an endothelium-specific JAM-A deficiency to DSS was similar to that of wild-type control mice, indicating that the response of mice with universal JAM-A deficiency was not solely due to loss of endothelial JAM-A89. unfortunately, the use of mice with a universal JAM-A deficiency and the presence of other epithelial cell abnormalities, particularly increased epi-thelial cell turnover, in the absence of exogenous stimuli limits interpretation of these studies. Even so, data from JAM-A-deficient mice do support the hypothesis that mucosal barrier loss can enhance the severity of colitis.

overall, data from both human subjects and mouse experimental models show that defects in tight junc-tion barrier function are insufficient to cause disease. However, several lines of evidence suggest that increased paracellular permeability can increase mucosal immune

activity, enhance disease progression and severity and, possibly, be a risk factor for development of disease. Finally, although much more investigation is needed, early reports indicate that restoration of tight junction barrier function may be effective, either alone or in combination with other agents, in preventing disease in at-risk individuals or maintaining remission in patients with inflammatory bowel disease.

Barrier loss activates immunoregulatory processes. why is tight junction barrier dysfunction alone insufficient to cause disease? Endoscopic mucosal resection, which removes the epithelium and mucosa completely and therefore causes barrier loss far greater than that caused by targeted tight junction dysfunction, is insufficient to cause chronic disease. Therefore, immuno regulatory mechanisms must be induced by the host following barrier loss to prevent inappropriate inflammatory responses, as mucosal damage is a daily occurrence in the gastrointestinal tract. Moreover, these immuno-regulatory mechanisms must be robust because, even in patients with inflammatory bowel disease, endoscopic mucosal resection is insufficient to initiate a relapse to active disease.

The increased transcription of IL-10 in the intestinal mucosa of mice with a constitutively active MLCK trans-gene may provide a clue to which immuno regulatory processes are triggered by barrier dysfunction. In addi-tion, responses to transient barrier loss have been used to explore mucosal immunoregulation92. In this model, intrarectal administration of ethanol caused transient epithelial cell damage, mucosal erosion and barrier loss. The induction of barrier loss was followed by an increase in the numbers of IFnγ- and IL-10-producing lamina propria mononuclear cells and lamina propria CD4+CD25+ T cells that express latency-associated peptide (LAP) on their surface92. Remarkably, such ethanol administration conferred protection from subsequent trinitrobenzene sulphonic acid (TnBS)-induced coli-tis, and this protection required the presence of LAP+ T cells92. The induction of these LAP+ T cells was shown to depend on CD11c+ DCs, Toll-like receptor 2 signalling and a normal luminal microorganism population92.

Although further study is needed to understand the mechanisms of LAP+ T cell induction by transient mucosal damage, it is interesting that in different experi-mental systems increased intestinal epithelial cell tight junction permeability increased the number of CD11c+ DCs in the superficial lamina propria86, and that inter-actions with intestinal epithelial cells enhance the abil-ity of bone marrow-derived CD11c+ DCs to induce the differentiation of regulatory T cells93. Together, these observations support the hypothesis that the interactions between CD11c+ DCs and luminal materials are regu-lated by tight junction permeability and are also central to mucosal immune homeostasis.

other data also support important roles for luminal material, particularly microorganisms and their prod-ucts, in mucosal immune regulation94. For example, antibiotics can be helpful in the management of Crohn’s disease95. Although the mechanisms by which antibiotics

R E V I E W S



http://www.uniprot.org/uniprot/Q9Y624



Epithelial cell

Laminapropria

Bacterial products and dietary antigens

MLCK

Endosome

Occludin

MHC

TCR

TReg cell

T cell TH1 cell

Antigen

IFNγTNF

TGFβRetinoic acid

IL-10 and TGFβ

IL-13

TH2 cell

Na+

Small solutes

Na+

Small solutes

Claudin Claudin-2

LAP

Actomyosinring

TLR

APC

promote maintenance of remission in patients with Crohn’s disease are not defined, they may be related to the observation that luminal microorganisms are required in experimental models of inflammatory bowel disease that include IL-10-deficient mice96,97 and adop-tive transfer colitis. Microorganisms are also necessary for the development of increased intestinal permea-bility before the onset of overt disease in IL-10-deficient mice76. Although this observation is not entirely under-stood, one interpretation is that the limited flux of microbial products that normally occurs across intact intestinal epithelial tight junctions is sufficient to trig-ger mucosal immune activation in IL-10-deficient mice. This mucosal immune activation could, in turn, result in the release of cytokines that cause increased intestinal permeability and accelerate disease progression (FIG. 3). However, in addition to immune cells, epithelial cells can respond to foreign materials. Lipopolysaccharide, for

example, enhances paracellular permeability in pulmo-nary and intestinal epithelium48,49. Conversely, epithelial Toll-like receptor 2 activation may restore barrier func-tion98. It will therefore be of interest to define the micro-bial products, dietary components and other factors that influence mucosal immune status following alterations in intestinal epithelial tight junction permeability.

Tight junctions integrate mucosal homeostasisone interpretation of the available data is that the tight junction barrier integrates the relationship between lumi-nal material and mucosal immune function (FIG. 3). In most individuals, this is a healthy relationship in which regulated increases in tight junction permeability or transient epithelial cell damage trigger the release of pro-inflammatory cytokines, such as TnF and IFnγ, as well as immunoregulatory responses. Immunoregulatory responses may include DC conditioning by epithelial cell-derived transforming growth factor-β (TGFβ) and retinoic acid, both of which can enhance regulatory T cell differentiation99,100. This precarious balance between pro-inflammatory and immunoregulatory responses can fail if there are exaggerated responses to pro-inflammatory cytokines, as may be associated with mutations in the endoplasmic reticulum stress response transcription factor X-box-binding protein 1 (XBP1) (ReF. 101), insuf-ficient IL-10 production (as in IL-10-deficient mice and, possibly, in patients with IL10 promoter polymor-phisms102) or inadequate immune tolerance to luminal antigens and microbial products94 (potentially because of NOD2 mutations in patients with inflammatory bowel disease103–106). As a result, mucosal immune cell activation may proceed unchecked and the release of cytokines, including TnF and IL-13, may enhance bar-rier loss that, in turn, allows further leakage of luminal material and perpetuates the pro-inflammatory cycle. This model highlights the roles of a susceptible host and defective epithelial cell barrier function as key compo-nents of the pathogenesis of intestinal inflammatory disease and explains the crucial role of the epithelial barrier in moulding mucosal immune responses.

ConclusionsSignificant progress has been made in understanding the processes by which physiological and pathophysio-logical stimuli, including cytokines, regulate the tight junction. Early reports suggest that restoration of tight junction barrier function may have benefit38,86. However, the mechanisms of tight junction regulation will have to be defined in greater detail if they are to be viable pharmacological targets. Conversely, recent data have emphasized the presence of immune mechanisms that maintain mucosal homeostasis despite barrier dysfunction, and some data suggest that the epithelium orchestrates these immunoregulatory events through direct interactions with innate immune cells. Future elucidation of the processes that integrate mucosal bar-rier function, or dysfunction, and immune regulation to prevent or perpetuate disease may lead to novel thera-peutic approaches for diseases associated with increased mucosal permeability.

Figure 3 | The epithelium and tight junction as integrators of mucosal homeostasis. Minor barrier defects allow bacterial products and dietary antigens to cross the epithelium and enter the lamina propria. This can lead to disease or homeostasis. If the foreign materials are taken up by antigen‑presenting cells (APCs), such as dendritic cells, that direct the differentiation of T helper 1 (T

H1) or T

H2 cells, disease can develop. In this

process, APCs and TH1 cells can release tumour necrosis factor (TNF) and interferon‑γ

(IFNγ), which signal to epithelial cells to increase flux across the tight junction leak pathway, thereby allowing further leakage of bacterial products and dietary antigens from the lumen into the lamina propria and amplifying the cycle of inflammation. This may, ultimately, culminate in established disease. Alternatively, interleukin‑13 (IL‑13) released by T

H2 cells increases flux across small cation‑selective pores, potentially contributing to

ongoing disease. Conversely, homeostasis may dominate if APCs promote regulatory T (T

Reg) cell differentiation, which can be enhanced by epithelial cell‑derived transforming

growth factor‑β (TGFβ) and retinoic acid. The TReg

cells display latency‑associated peptide (LAP) on their surfaces and may secrete IL‑10 and TGFβ to prevent disease. MLCK, myosin light chain kinase; TLR, Toll‑like receptor; TCR, T cell receptor.

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AcknowledgementsI am indebted to current and past members of my laboratory for discussions and investigations that contributed to this article. Thanks also to A. M. Marchiando and C. R. Weber for the images used in figures 1 and 2. Work in my laboratory is currently supported by the US National Institutes of Health (DK061931, DK068271, DK67887 and HL091889), the University of Chicago Cancer Center (CA14599), the University of Chicago Digestive Disease Research Core Center (DK042086 ) , t he US Depar tmen t o f De f ense (W81XWH-09-1-0341), the Broad Medical Research Program (IBD-0272) and the Chicago Biomedical Consortium. I apologize to colleagues whose work and publications could not be referenced owing to space constraints.

DATABASESUniProtKB: http://www.uniprot.orgα‑catenin 1 | β‑catenin | catenin δ1 | claudin‑1 | claudin‑2 | claudin‑16 | E‑cadherin | IFNγ | IL‑1β | IL‑10 | JAM‑A | LAP | LIGHT | MLCK | NHE3 | TNF | XBP1 | ZO1 | ZO2

FURTHER INFORMATIONJerrold R. Turner’s homepage: http://pathologyweb.bsd.uchicago.edu/faculty/JTurner.html

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